1. Introduction

Conventional wide-field and confocal scanning optical microscopy techniques enable fluorescent molecules to be imaged with a spatial resolution of approximately one half of the incident wavelength. Recently, however, scanning probe microscopy techniques such as atomic force microscopy, scanning tunneling microscopy, and near-field scanning optical microscopy have been developed which provide a nano-scale spatial resolution to break the optical diffraction limit. Furthermore, optical distance measurements of less than 10 nm can now be achieved by evaluating the fluorescent resonance energy transfer between a fluorescent donor and an acceptor. However, not all of these approaches are compatible with the observation of live cells [1]. Experimental evidence suggests that the molecular interactions which occur on or near the surfaces of cell membranes (typically within 50 nm) have different properties from those which occur in the bulk solution. To visualize these events within the cell-substrate contact region more clearly, it is desirable to use observation techniques which suppress the interference arising from background fluorescence [2]. In total internal reflection fluorescence microscopy (TIRFM), an incident light ray with an incident angle greater than the critical angle is used to generate an evanescent field to excite and image fluorophores on or very near (i.e. within 100 nm) the liquid/solid interface. Fluorophores located within or close to the plasma membrane are excited by the shallow evanescent field, resulting in images with very low background fluorescence and no out-of-focus fluorescence. TIRFM is particularly suited to the investigation of live cells since it causes minimal photodamage or photobleaching. Furthermore, since TIRFM typically illuminates a wide-field thinner vertical slice compared to confocal scanning systems, the signal-to-noise ratio (SNR) and attainable frame rate are significantly improved [3,4]. Although confocal microscopes can generate deep three-dimensional images, TIRFM provides a complementary approach which can be readily combined with other microscopy techniques [5]. Therefore, TIRFM has been widely used in studies of cell-substrate contact regions [6], protein dynamics [7], endocytosis or exocytosis [8], and membrane-associated photosensitizers [9].

Some improvements in TIRFM have been made regarding its experimental configuration and image processing techniques. However, the fluorescence signal in live cell imaging still needs enhancement, if dynamic images of the molecular interactions which take place on or near the surface of the cell membrane are to be acquired. As of this point, even if given a readily detectable fluorescence signal of single molecules, reduced detection limits are still required [10]. In recent investigations, metallic surfaces or particles has been exploited to enhance the fluorescence of the fluorophores in order to develop more efficient fluorescence immunoassays [11–14]. Previous studies have also presented cellular-based diagnostic techniques based on metallic nanoparticle scattering [15,16]. In these techniques, surface plasmons (SPs) or localized SPs on the metallic surfaces or nanoparticles enhance the local electro-magnetic (EM) field around the fluorophore and therefore increase the intensity of the detected fluorescence signal [14]. SPs are oscillations of the free electrons located on the surface of a metal film and can be excited by incident light using the attenuated total reflection (ATR) method. When the phase velocity of an incident evanescent p-polarization light ray matches that of the SPs, the so-called surface plasmon resonance (SPR) phenomenon occurs and the SPR associated with the EM field is greatly enhanced [17]. It has been reported that the localized SPs provided by metal nanoparticles or sub-wavelength metal structures can enhance the EM field by a factor of at least 10 times compared to that of SPs [18]. However, live cells will not readily adhere or migrate to such metal nanostructures. In order to simply image the phenomenon of live cells interacting with the biosurface, the localized SPs configuration is therefore not adopted in this study. In the Kretschmann SPR configuration, the ATR method is employed to excite the SPs on a thin metal film deposited on the surface of the base of a prism [19]. If the metal film is thin enough that the EM fields of the nonradiative SPs contact the prism medium, then their transformation into light is possible [17]. This light from the emission SPs can be referred to as the back-scattered field. In the interaction of fluorophores with SPs over a very short metal-fluorophore distance d, the dipole field is dominated by the near field and the strength of the fluorescence decreases as d ^-4 [10]. In other words, the fluorescence is ‘quenched’ and the excitation energy has dissipated into the metal in the form of heat with a very short metal-fluorophore distance (approximately d<10 nm) [20–22]. For intermediate separation distances, the emission fluorescence can be effectively coupled back to the SPs of the metal surface by matching the momentum of the fluorophores with that of the SPs. Subsequently, the emission SPs are re-radiated into the glass prism with a hollow cone of intense light around angles near the SPR angle [23]. Efficient single-molecule fluorescence experiments through a thin metal film are possible [24]. In one previous study, however, it was shown that the surface-plasmon coupled emission does not lead to any improvement, but the metal film actually reduces the sensitivity of fluorescence detection [25]. The results related to the fluorescence rate are still not consistent. For larger separation distances, the fluorescent emission rate can be predicted by considering the classical picture of a dipole, which is influenced by the back-reflected field caused by its dipole image [26]. Therefore, through the moderate modification of a metal film or particles on a substrate and the metal-fluorophore distance, the plasmonic effect is the observed increase in the quantum yield and photostability, the reduction in the fluorescence lifetime, and the specific orientation in the typically isotropic emission. [10,27,28]. These effects are not due to the reflection of the emitted photons, but rather as the result of the fluorophore dipole interacting with free electrons in the metal [28].

Previously reported surface plasmon-enhanced fluorescence measurement techniques were designed only for single-spot sensing applications. However, this study proposes a surface plasmon-enhanced fluorescence measurement technique capable of imaging the molecular interactions between a cell membrane and a biosurface in real-time. Thrombomodulin (TM), an integral membrane glycoprotein, is a thrombin receptor identified originally on the endothelium which acts as a natural anticoagulant [29]. Recent investigations have supported the contention that TM plays an important role in extravascular activities [30] and TM-mediated cell adhesion [31]. Using membrane protein TM fused to green fluorescent protein (GFP) to study the prevention of cancer formation is a crucial research topic in the cell biology field [32]. Therefore, this study employs the proposed surface plasmon-enhanced TIRFM technique to dynamically image the interaction of membrane protein TM with a collagen biosurface. The experimental results demonstrate that the live cell membrane images produced by the surface plasmon-enhanced TIRFM technique are approximately one order of magnitude brighter than those of the conventional TIRFM approach with an equivalent image quality.

2. Materials and methods

2.1 Optical system setup

Figure 1(a) shows the optical configuration of the proposed prism-coupled surface plasmon-enhanced TIRFM, in which the ATR method is used to excite the SPs in order to enhance the local EM field and to increase the SNR of the fluorescence. In this configuration, a thin silver film via a sputtering deposition process, a chemical self-assembly monolayer (SAM), and a biomolecular layer are sequentially deposited on an SF-11 slide in accordance with an optimal design which is known to enhance the intensity of the fluorescence and therefore to increase the attainable frame rate. As shown in Fig. 1(a), a light beam from a diode-pumped solid-state laser (20 mW, λ=473 nm) is passed initially through two polarizers, which enable the intensity and plane of polarization of the laser beam to be adjusted such that the light beam which exits the polarizers contains a higher ratio of p-polarization wave than s-polarization wave. The light beam is then focused by a convergence lens (f=175 mm), passes through a coupled SF-11 hemispherical prism, a layer of index-matching oil, and the SF-11 slide, and then incidents directly upon the interface between the slide and the thin silver film. Using the hemispherical prism, the angle of the incident light can be easily adjusted in order to vary the penetration depth of the evanescent field into the thin silver film (thickness 50.0±1.0 nm). In the current Kretschmann configuration, an SF-11 hemispherical prism with a refractive index of 1.811@473 nm is used to increase the wave vector of the incident light and to reduce the SPR angle, θ _spr , i.e., the angular position of the dip in the reflectivity spectrum. Fluorescence from the cell membrane excited by the SPs or the evanescent wave is collected and imaged from the reverse side of the prism by an immersion water objective (100×, N.A.=1.0, Olympus) dipped into the window of the flow cell, and is then passed through a band-pass filter (BPF, λ=500~535 nm, Semrock) into a high-speed frame rate CCD camera (iXon DV885, Andor).

2.2 Cell culture protocol

Thrombomodulin (TM), purified from human lung endothelial membrane preparations, has an apparent molecular weight of 78 kDa and consists of 557 amino acid residues arranged in five distinct domains [33]. In this study, cultured human melanoma cells were stably transfected with DNA plasmids encoded with the target protein TM fused to GFP. The human melanoma-GFP-tagged TM cells were maintained in a Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 0.292 g/liter L-glutamine, 2% sodium bicarbonate, 1% sodium pyruvate, 1% penicillin, and 10% fetal bovine serum. To maintain the live cells stably expressing TM-GFP fusion proteins, the cells were cultured in DMEM supplemented with 300 µg/ml antibiotic G418. When the dish was one-eighth to one-seventh full of adhered cells, the culture media were removed from the dish. The live cell monolayer was washed with a phosphate buffer solution (pH 7.4) containing no Ca²⁺/Mg²⁺, and 1 ml trypsin/EDTA was then added to the dish to cover the cell monolayer. The culture dish was then placed into an incubator and left for 2~5 minutes. An inverted microscope (Axiovert 200, Carl Zeiss) was then used to check that all the cells were detached.

The cells were then re-suspended in a small volume of fresh DMEM. In order to compare the fluorescence intensities of a conventional total internal reflection (TIR) sensing chip with that of the proposed enhanced SPR chip, the required number of cells (approximately 10⁶ cells/cm²) were added to collagens immobilized by chemical SAMs on a naked SF-11 slide and on a silver thin film slide containing pre-warmed medium, respectively. Figure 1(b) shows a cell cultured on a collagen-coated slide modified with a silane SAM. In fabricating this conventional TIR chip, the naked SF-11 slide was immersed in 20% (3-aminoproply)triethoxysilane solution in order to form a dense SAM on its surface. To immobilize the protein collagen, covalent activation was conducted by immersing the chip in a solution containing EDC[N-(3-dimethylaminopropyl)-N’-ethylcarbodimide hydrochloride, 2 mM] and NHS(N-hydroxysuccinimide, 5 mM) for 6 hours. In Fig. 1(c), the cell is cultured on a collagen-coated silver thin film modified with a thiol SAM. In developing this enhanced SPR chip, the metal film was immersed in 1 mM 2-aminoethanethiol hydrochloride solution to form a dense SAM on its surface. As with the TIR chip, covalent activation was then performed by immersing the chip in a solution containing EDC[N-(3-dimethylaminopropyl)-N’-ethylcarbodimide hydrochloride, 2 mM] and NHS(N-hydroxysuccinimide, 5 mM) for 6 hours to immobilize the protein collagen. One prior study for detecting fluorescent erythrocytes on metal surfaces to study the quenching effect with a similar optical system setup and cell culture protocol can be referred to in [34].

2.3 Excitation of surface plasmons by incident light

SPR is an optical phenomenon in which incident p-polarization light excites SPs, i.e. a surface charge density oscillation, at the interface between a thin metal film and a dielectric sample medium. SPR occurs when the parallel component of the wave vector of the incident p-polarization light, k _x , matches the wave vector of the SPW traveling over a semi-infinite structure, $k_{\text{sp}}^0$, i.e.

 

Fig. 1. (a) Schematic illustration of experimental configuration employed for live cell imaging using conventional and surface plasmon-enhanced TIRFM techniques. (b) Conventional TIR chip: cell is cultured on collagen-coated slide modified with silane. (c) Enhanced SPR chip, cell is cultured on collagen-coated silver thin film modified with thiol.

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where ε ₀, ε ₁, and ε ₂ are the wavelength-dependent complex dielectric constants of the coupled-prism, the metal layer, and the dielectric sample, respectively, and θ denotes the SPR angle, θ _spr . As shown in Fig. 1(c), the SPR optical configuration basically consists of a prism, a thin metal film, and an analytic layer on which immobilized analytes are used to probe the target receptors [18]. The SPR angle depends on the dielectric constant and thickness of the nanolayer beneath the sensing metal layer. Simplifying the SPR configuration to the current three-layer 0/1/2 system, the optical coupling of the SPR sensor can be analyzed by calculating the reflectivity R ₀₁₂ using the Fresnel equation, i.e.

where ε _i and k _zi =[ε _i (ω/c)²-$k_x^2$ ]^1/2 are the dielectric constants and the wave vector components perpendicular to the interface in medium i, respectively, and d ₁ is the finite thickness of the metal film.

Figure 2(a) plots the reflectivity spectrum of the TIR chip as a function of the incident angle. In this figure, the solid line represents the simulated reflectivity spectrum, while the dashed line indicates the enhancement factor of the electric field intensity at the interface between the collagen and the buffer. In the TIR chip, the thicknesses of the collagen and silane layers are approximately 5.5 nm and 1.0 nm, respectively, and the corresponding refractive indexes are 1.46 and 1.44. Figure 2(a) shows that the maximum enhancement factor of the electric field at the interface between the collagen and the buffer is 7.31 and occurs at a critical angle of 47.56°. Meanwhile, Figure 2(b) shows that the distribution of the enhancement factor of the electric field intensity is perpendicular to the interface of the TIR chip at an angle of 57.46°. The maximum value of the enhancement factor is 3.62. Figure 3(a) shows the simulated reflectivity spectrum (solid line) and the enhancement factor of the electric field intensity at the interface between the collagen and the buffer (dashed line) in the SPR chip. The thickness and refractive index of the thiol layer are 1.1 nm and 1.49, respectively. In Fig. 3(a), the angle associated with the maximum enhancement of the electric field intensity, i.e. 57.46°, is slightly lower than the SPR angle at 57.58°. Figure 3(b) shows that the distribution of the enhancement factor of the electric field intensity is perpendicular to the interface of the SPR sensing chip at 57.46°. The enhancement factor at the interface between the collagen and the buffer has a value of 75.61. In other words, the local field enhancement of the enhanced SPR chip (75.61) is theoretically 20.87 times higher than that of the conventional TIR chip (3.62). Local field enhancement leads to an increased excitation rate, and is therefore expected to enhance the fluorescence intensity. However, the interaction of fluorophores with SPs over a short metal-fluorophore distance is happened, and hence the fluorescence quenching effect is counted [22]. Taking the competing phenomena into account, the enhancement in the fluorescence signal obtained from the proposed SPR chip is more likely to be no more than 20.87 times that of the TIR chip. To compensate for the two competing effects, a spacer is inserted (a thiol SAM and a collagen layer in this study) to increase the distance between the live cell membrane and the metal film such that the experimental results demonstrate a capability of producing 10-times brighter fluorescent live cell imaging via the SPs.

3. Experimental results and discussions

Figures 4(a) and 4(b) present conventional TIRFM images of melanoma-GFP-tagged TM cells (measuring approximately 30 × 30 µm²) cultured on collagen-coated slides modified with silane. The exposure times of the images in Figs. 4(a) and 4(b) are 0.5 seconds and 0.04 seconds, respectively, and the corresponding fluorescence intensity scales are 500 and 50 (arbitrary unit, a.u.), respectively. Figures 4(c) and 4(d) show surface plasmon-enhanced TIRFM images of the melanoma-GFP-tagged TM cells cultured on a collagen-coated silver thin film modified with thiol. The exposure times of Figs. 4(c) and 4(d) are again 0.5 seconds and 0.04 seconds, respectively. However, in these images, the corresponding fluorescence

intensity scales are 5,000 and 500, respectively. Comparing Fig. 4(a) and Fig. 4(c), both with an exposure time of 0.5 seconds, it is apparent that the surface plasmon-enhanced fluorescent cell image (Fig. 4(c)) is approximately 10 times brighter than the conventional TIRFM cell image (Fig. 4(a)). Furthermore, it can be seen that the surface plasmon-enhanced fluorescent cell image shown in Fig. 4(d), with a reduced exposure time of 0.04 seconds, has a higher signal-to-noise (SNR) ratio (i.e. a clearer image) than the corresponding conventional TIRFM cell image shown in Fig. 4(b). Significantly, comparing Figs. 4(a) and 4(d), it is apparent that the fluorescence intensity and SNR of the image acquired using the surface plasmon-enhanced TIRFM technique (Fig. 4(d)) are comparable to those of the image acquired using conventional TIRFM even though the exposure time of the enhanced technique is only approximately one-tenth that used in the conventional approach. The greater the fluorescence intensity and the higher the SNR value of the image, the shorter the required exposure time and hence the faster the frame rate which can be achieved. Therefore, the results of Fig. 4 indicate that the proposed surface plasmon-enhanced TIRFM technique improves both the brightness of the cell images and the frame rate at which they can be acquired by approximately one order of magnitude. Figure 4(e) plots the fluorescence intensity versus the X-axis position (µm) along the central crosscut lines on the live cell membrane images in Figs. 4(a) and 4(c). Note that the dashed line corresponds to the conventional TIRFM image (Fig. 4(a)), while the solid line corresponds to the surface plasmon-enhanced TIRFM image (Fig. 4(c)). It can be seen that the fluorescence intensity is increased by approximately one order of magnitude by the SPs in the enhanced TIRFM chip compared to that of the chip based on evanescent wave excitation only.

 

Fig. 2. (a) Solid line: reflectivity spectrum of TIR chip as function of incident angle; Dashed line: enhancement factor of electric field intensity at interface between collagen and buffer. (b) Enhancement factor distribution is perpendicular at sensing interface at incident angle of 57.46°.

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Fig. 3. (a) Solid line: reflectivity spectrum of SPR chip as function of incident angle; Dashed line: enhancement factor of electric field intensity at interface between collagen and buffer. (b) Enhancement factor distribution is perpendicular at sensing interface at incident angle of 57.46°.

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The simulation results presented in Fig. 3 suggest that a twenty-fold enhancement of the fluorescence intensity at the interface between the collagen and the buffer can be obtained in the surface plasmon-enhanced TIRFM sensing chip. However, the experimental results presented in Fig. 4 show that the fluorescence enhancement is only one order of magnitude in practice. Recent studies have shown that the fluorescence intensity of fluorophores close to the metal surface reduces significantly as a result of fluorescence quenching, in which a non-radiative resonance energy transfer (RET) into the metal film occurs, causing the excitation energy to be dissipated in the metal in the form of heat [21,22]. As a result of the distance-dependent nature of the RET phenomenon, the surface plasmon-enhanced fluorescence signal shows distance-dependent characteristics, and the quenching efficiency therefore varies as a function of the distance between the metal film and the fluorophores. Specifically, the greater the separation of the fluorophores from the metal film, the smaller the quenching efficiency [35]. In the surface plasmon-enhanced TIRFM sensing chip developed in this study, the non-absorbing dielectric layer consisting of collagen and thiol layers serves as a spacer of thickness 6.1 nm between the silver thin film and the fluorophore-containing cell membrane. Hence, the fluorescence quenching phenomenon is reduced somewhat. In other words, when cells adhere to the collagen layer, the fluorescence intensity signal is enhanced by the SP effect, but is simultaneously reduced by the fluorescence quenching effect. The experimental results indicate that the collagen/thiol spacer between the metal film and the live cell membrane enables a ten-fold improvement in the image intensity and acquisition rate of the surface plasmon-enhanced TIRFM technique compared to the conventional TIRFM approach.

 

Fig. 4. TIRFM live cell membrane images exposed at (a) 0.5 seconds and (b) 0.04 seconds; Surface plasmon-enhanced TIRFM images exposed at (c) 0.5 seconds and (d) 0.04 seconds; (e) Distribution of fluorescence intensity along x-axis of central crosscut lines of (a) conventional TIRFM live cell membrane image (dashed line) and (c) Surface plasmon-enhanced TIRFM image (solid line).

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Moreover, the detection of a fluorophore is usually limited by its quantum yield and photostability [10]. The fluorescence intensity (quantum yield) is not only determined by the intensity of the excitation SP field but also by the quenching influence of the molecular dipole-metal interaction [21]. In this study, a spacer that is 6.1 nm thick is placed between the silver thin film and the fluorophore-containing cell membrane and is used to reduce the effect of metallic surface-induced fluorophore quenching at very short distances (approximately 0~5 nm) [10,26]. However, the key issue in fluorescence microscopy is not merely how to increase the quantum yield, but rather how to collect as many photons from fluorophore as possible before photobleaching occurs. Although the quenching effect reduces the fluorescence emission, it shortens its lifetime to enhance fluorophore photostability [28]. Therefore, the quantum yield and photostability of fluorophores should be modified and controlled in order to improve the detection limit [10,28]. This study has demonstrated that the proximity to metallic surfaces > 5 nm can increase the local EM field (or concentrate the incident field) about ten times. In contrast to the relatively constant radiative decay rate via an increase of incident laser power, the radiative decay rate can be increased by placing the fluorophores at suitable distances from metallic surfaces. An increase in radiative decay rate results in an increased fluorescence intensity and a reduction in lifetime [22,27]. Therefore, the fluorescence intensity is not only enhanced by the excitation SP field, but also the fluorophore photostability which can be improved with metal coatings.

4. Conclusions

This study has developed a surface plasmon-enhanced TIRFM imaging technique for the real-time investigation of the interactions of live cells on a biomolecular surface. The developed technique has been successfully employed to observe the interactions of melanoma-GFP-tagged TM cells with a collagen biosurface. The results have shown that compared to the conventional TIRFM imaging technique, which is based on evanescent wave excitation only, the additional excitation provided by the SPs in the enhanced TIRFM technique, increases the brightness and frame rate acquisition of live cell membrane images by approximately one order of magnitude while retaining an equivalent image quality.